Retrovirus vectors are a common tool for gene delivery (Miller, Nature (1992) 357:455-460). The biology of retroviral proliferation enables such a use. Typically, wild type full length retroviral mRNA's serve both as a template for synthesis of viral proteins and as the viral genome. Such mRNA's encompass a region called the encapsidation signal which binds certain viral proteins thereby ensuring specific association of that mRNA with the produced virions. On infection of the target cell, reverse transcription of the retroviral mRNA into double stranded proviral DNA occurs. The retroviral enzyme, integrase, then, binds to both long terminal repeats (LTR) which flank the proviral DNA and subsequently catalyzes the integration thereof into the genomic DNA of the target cell. Integrated proviral DNA serves as the template for generation of new full-length retroviral mRNA's.
Retroviral vectors have been tested and found to be suitable delivery vehicles for the stable introduction of a variety of genes of interest into the genomic DNA of a broad range of target cells, a process known as transduction of the cells with the gene of interest. The ability of retrovirus vectors to deliver an unrearranged, single copy gene into a broad range of, for example, rodent, primate and human somatic cells makes retroviral vectors well suited for transferring genes to a cell.
A primary approach in retrovirus-derived vector design relies on removal of the encapsidation signal and sequences coding the LTR's from the viral genome without affecting viral protein expression and transfer of such sequences to the construct including a nucleic acid coding the gene of interest, sometimes called the transfer vector.
A useful adjunct for producing recombinant retroviral vectors are packaging cell lines which supply in trans the proteins necessary for producing infectious virions, but those cells are incapable of packaging endogenous viral genomic nucleic acids (Watanabe & Temin, Molec. Cell. Biol. (1983) 3:2241-2249; Mann et al., Cell (1983) 33:153-159; and Embretson & Temin, J. Virol. (1987) 61:2675-2683). Expression in the vector producer cells of both viral core proteins, which comprise the virion particle, and mRNA containing LTR, encapsidation sequences and the gene of interest, results in release by the cells of particles which phenotypically resemble parental retrovirus, but carry the gene of interest instead of the viral genome. Such particles will integrate the gene of interest but not the viral DNA into the genome of target cells.
A consideration in the construction of retroviral packaging cell lines is the production of high titer vector supernatants free of recombinant replication competent retrovirus (RCR), which have been shown to produce T cell lymphomas in rodents (Cloyd et al., J. Exp. Med. (1980) 151:542-552) and in primates (Donahue et al., J. Exp. Med. (1992) 176:1125-1135).
In the vector producing cells, restoration of the physical association of LTR and encapsidation sequences with the sequences coding the viral proteins may lead to the emergence of RCR capable of self amplification. Generation of recombinant viruses during vector production is highly undesirable for several reasons. First, the recombinant mRNA may compete with the transgene mRNA for encapsidation into virions thereby decreasing the number of transgenes per vector particle made by producer cells. That competition, as well as amplification of such recombinants in producer cells, may lead to the exponential loss of vector transduction potential.
Second, such recombinants, if undetected during vector production, may be introduced unintentionally to the vector recipients. There, transfer of the recombinant genome to the host may cause otherwise avoidable toxicity or an immune reaction to the transduced cells. Importantly, viral recombinants may be pathogenic or may evolve into pathogens on additional rounds of amplification and/or through additional events of recombination with endogenous sequences of the host cells (such as endogenous retroviral sequences).
Recombinant retrovirus could be generated at the DNA or mRNA level. DNA recombination may take place if plasmid constructs independently coding for packaging and transfer vector functions are mixed and co-transfected in an attempt to create transient producer cells. To decrease the chance of recombination at the DNA level, the constructs could be introduced into cells one after another with concurrent selection of clones after each construct is associated stably with the cellular genome. Somatic cells dividing mitotically generally do not undergo crossing over between homologous chromosomes and since each vector construct association is expected to be integrated randomly into the genomic DNA, the likelihood of close association and therefore the chance of recombination is low.
Recombination at the mRNA level may take place during reverse transcription when both packaging mRNA and transfer vector mRNA (even when generated by separated expression constructs) become co-encapsidated into viral particles. The retroviral enzyme reverse transcriptase (RT) uses mRNA as template for DNA synthesis. Also, RT is known to switch between or away templates. Thus, if two different mRNA's are present within a viral particle, when combined, a single DNA unit could be synthesized by the RT as the result of template switching.
One approach to minimize the likelihood of generating RCR in packaging cells is to divide the packaging functions into two or more genomes, for example, one which expresses the gag and pol gene products and the other which expresses the env gene product (Bosselman et al., Molec. Cell. Biol. (1987) 7:1797-1806; Markowitz et al., J. Virol. (1988) 62:1120-1124; and Danos & Mulligan, Proc. Natl. Acad. Sci. (1988) 85:6460-6464). That approach minimizes the ability for co-packaging and subsequent transfer of the two or more genomes, as well as significantly decreasing the frequency of recombination due to the presence of multiple retroviral genomes in the packaging cell to produce RCR.
The rationale behind the approach of splitting the packaging functions is that multiple recombination events must occur to generate RCR. That approach, however, does not decrease the chance of individual recombination events. Therefore partial recombinants incapable of amplification could be generated. To monitor emergence of such partial recombinants, novel complementing detection systems must be designed.
In the event recombinants arise, mutations (Danos & Mulligan, supra) or deletions (Boselman et al., supra; and Markowitz et al., supra) within vector constructs can be configured such that in the event recombinants arise, those will be rendered non-functional. In addition, deletion of the 3′ LTR on both packaging constructs further reduces the ability to form functional recombinants.
It was demonstrated previously for many biological systems that the frequency of recombination between two genetic elements is directly proportional to the extent of homologous sequences. Thus, another approach is to minimize the extent of sequence homology between and amongst the vectors. Technical difficulties associated with minimization of the homologous sequences between transfer vector and packaging constructs can be explained by the fact that some essential genetic elements could not be removed from at least one of the constructs without significant loss of transduction potential.
Lentiviruses are complex retroviruses which, in addition to the common retroviral genes gag, pol and env, contain other genes with regulatory or structural function. The higher complexity enables the lentivirus to modulate the life cycle thereof, as in the course of latent infection.
Lentiviruses have attracted the attention of gene therapy investigators because of the ability to integrate into non-dividing cells (Lewis et al., EMBO J. (1992) 11:3053-3058; Bukrinsky et al., Nature (1993) 365:666-669; Gallay et al., Proc. Natl. Acad. Sci. USA (1997) 94:9825-9830; Gallay et al., Cell (1995) 80:379-388; and Lewis et al., J. Virol. (1994) 68:510). Replication-defective vectors from the human lentivirus human immunodeficiency virus (HIV) transduce target cells independent of mitosis (Naldini et al., Science (1996) 272:263-267). The vectors proved highly efficient for in vivo gene delivery and achieved stable long-term expression of the transgene in several target tissues, such as the brain (Naldini et al., PNAS (1996) 93:11382-1138; and Blomer et al., J. Virol. (1997) 71:6641-6649), the retina (Miyoshi et al., PNAS (1997) 94:10319-10323), the liver and the muscle (Kafri et al., Nature Genetics (1997) 17:314-317).
A typical lentivirus is HIV, the etiologic agent of AIDS. In vivo, HIV can infect terminally differentiated cells that rarely divide, such as lymphocytes and macrophages. In vitro, HIV can infect primary cultures of monocyte-derived macrophages (MDM) as well as HeLa-Cd4 or T lymphoid cells arrested in the cell cycle by treatment with aphidicolin or γ irradiation.
The complexity of the lentiviral genome may be exploited to build novel biosafety features in the design of a retroviral vector. In addition to the structural gag, pol and env genes common to all retroviruses, HIV contains two regulatory genes, tat and rev, essential for viral replication, and four accessory genes, vif, vpr, vpu and nef, that are not crucial for viral growth in vitro but are critical for in vivo replication and pathogenesis (Luciw., in Fields et al. (ed.), “Fields Virology”, 3rd ed., (1996) p. 1881-1975 Lippincott-Raven Publishers, Philadelphia.).
The Tat and Rev proteins regulate the levels of HIV gene expression at transcriptional and post-transcriptional levels, respectively. Due to the weak basal transcriptional activity of the HIV LTR, expression of the provirus initially results in small amounts of multiply spliced transcripts coding for the Tat, Rev and Nef proteins. Tat dramatically increases HIV transcription by binding to a stem-loop structure (TAR) in the nascent RNA thereby recruiting a cyclin-kinase complex that stimulates transcriptional elongation by the polymerase II complex (Wei et al., Cell (1998) 92:451-462)). Once Rev reaches a threshold concentration, Rev promotes the cytoplasmic accumulation of unspliced and singly-spliced viral transcripts leading to the production of the late viral proteins.
Rev accomplishes that effect by serving as a connector between an RNA motif (the Rev-responsive element, RRE) found in the envelope coding region of the HIV transcript and components of the cell nuclear export machinery. Only in the presence of Tat and Rev are the HIV structural genes expressed and new viral particles produced (Luciw, supra).
In a first generation of HIV-derived vectors (Naldini et al., Science, supra), viral particles were generated by expressing the HIV-1 core proteins, enzymes and accessory factors from heterologous transcriptional signals and the envelope of another virus, most often the G protein of the vesicular stomatitis virus (VSV.G; Burns et al., PNAS (1993) 90:8033-8037) from a separate plasmid.
In a second version of the system, the HIV-derived packaging component was reduced to the gag, pol, tat and rev genes of HIV-1 (Zufferey et al., Nat. Biotech. (1997) 15:871-875).
In either case, the vector itself carried the HIV-derived cis-acting sequences necessary for transcription, encapsidation, reverse transcription and integration (Aldovini & Young., J. Virol. (1990) 64:1920-1926; Berkowitz et al., Virology (1995) 212:718-723, Kaye et al., J. Virol. (1995) 69:6588-6592; Lever et al., J. Virol. (1994) 63: 4085-4087; McBride et al., J. Virol. (1989) 70:2963-2973; McBride et al., J. Virol. (1997) 71:4544-4554; Naldini et al., Science (supra); and Parolin et al., J. Virol. (1994) 68: 3888-3895).
Such a vector thus encompassed from the 5′ to 3′ end, the HIV 5′ LTR, the leader sequence and the 5′ splice donor site, approximately 360 base pairs of the gag gene (with the gag reading frame closed by a synthetic stop codon), 700 base pairs of the env gene containing the RRE and a splice acceptor site, an internal promoter, for example, typically the immediate early enhancer/promoter of human cytomegalovirus (CMV) or that of the phosphoglycerokinase gene (PGK), the transgene and the HIV 3′ LTR. Vector particles are produced by co-transfection of the constructs in 293T cells (Naldini et al., Science, supra). In that design, significant levels of transcription from the vector LTR and of accumulation of unspliced genomic RNA occur only in the presence of Tat and Rev.
Infection of cells is dependent on the active nuclear import of HIV preintegration complexes through the nuclear pores of the target cells. That occurs by the interaction of multiple, partly redundant, molecular determinants in the complex with the nuclear import machinery of the target cell. Identified determinants include a functional nuclear localization signal (NLS) in the gag matrix (MA) protein, the karyophilic virion-associated protein, vpr, and a C-terminal phosphotyrosine residue in the gag MA protein.